Differential sensing with BioFET sensors

ABSTRACT

A sensor array includes a semiconductor substrate, a first plurality of FET sensors and a second plurality of FET sensors. Each of the FET sensors includes a channel region between a source and a drain region in the semiconductor substrate and underlying a gate structure disposed on a first side of the channel region, and a dielectric layer disposed on a second side of the channel region opposite from the first side of the channel region. A first plurality of capture reagents is coupled to the dielectric layer over the channel region of the first plurality of FET sensors, and a second plurality of capture reagents is coupled to the dielectric layer over the channel region of the second plurality of FET sensors. The second plurality of capture reagents is different from the first plurality of capture reagents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/737,645, titled “Microorganism Screening and CultureBioFET Chip,” filed Sep. 27, 2018, the disclosure of which isincorporated by reference herein in its entirety.

BACKGROUND

Biosensors are devices for sensing and detecting biomolecules andoperate on the basis of electronic, electrochemical, optical, andmechanical detection principles. Biosensors that include transistors aresensors that electrically sense charges, photons, and mechanicalproperties of bio-entities or biomolecules. The detection can beperformed by detecting the bio-entities or biomolecules themselves, orthrough interaction and reaction between specified reactants andbio-entities/biomolecules. Such biosensors can be manufactured usingsemiconductor processes, can quickly convert electric signals, and canbe easily applied to integrated circuits (ICs) andmicroelectromechanical systems (MEMS).

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the followingdetailed description when read with the accompanying figures. It isnoted that, in accordance with the standard practice in the industry,various features are not drawn to scale. In fact, the dimensions of thevarious features may be arbitrarily increased or reduced for clarity ofdiscussion.

FIG. 1 illustrates components of a sensing device, according to someembodiments.

FIG. 2 illustrates a cross-sectional view of an exemplary dual-gateback-side sensing FET sensor, according to some embodiments.

FIG. 3 is a circuit diagram of a plurality of FET sensors configured inan exemplary addressable array, according to some embodiments.

FIG. 4 is a circuit diagram of an exemplary addressable array of dualgate FET sensors and heaters, according to some embodiments.

FIG. 5 illustrates a cross-sectional view of an exemplary dual gateback-side sensing FET sensor, according to some embodiments.

FIGS. 6A and 6B illustrate using the dual gate back-side sensing FETsensor as a pH sensor, according to some embodiments.

FIG. 7 illustrates a cross-sectional view of an exemplary dual gateback-side sensing bioFET detecting the presence of cells or othermicroorganisms, according to some embodiments.

FIG. 8 illustrates an example metabolic pathway for glucose.

FIG. 9 illustrates a sensing array of FET sensors, according to someembodiments.

FIG. 10 illustrates capturing analytes using a sensing array, accordingto some embodiments.

FIG. 11 illustrates capturing different analytes using a sensing array,according to some embodiments.

FIG. 12 illustrates monitoring cell growth using a sensing array,according to some embodiments.

FIG. 13 illustrates disposing different capture reagents across asensing array, according to some embodiments.

FIG. 14 illustrates a flowchart of an example method for performingsensing with a sensor array.

FIG. 15 illustrates a flowchart of another example method for performingsensing with a sensor array.

DETAILED DESCRIPTION

The following disclosure provides many different embodiments, orexamples, for implementing different features of the provided subjectmatter. Specific examples of components and arrangements are describedbelow to simplify the present disclosure. These are, of course, merelyexamples and are not intended to be limiting. For example, the formationof a first feature over a second feature in the description that followsmay include embodiments in which the first and second features areformed in direct contact, and may also include embodiments in whichadditional features may be formed and/or disposed between the first andsecond features, such that the first and second features may not be indirect contact. In addition, the present disclosure may repeat referencenumerals and/or letters in the various examples. This does not in itselfdictate a relationship between the various embodiments and/orconfigurations discussed.

Further, spatially relative terms, such as “beneath,” “below,” “lower,”“above,” “upper” and the like, may be used herein for ease ofdescription to describe one element or feature's relationship to anotherelement(s) or feature(s) as illustrated in the figures. The spatiallyrelative terms are intended to encompass different orientations of thedevice in use or operation in addition to the orientation depicted inthe figures. The apparatus may be otherwise oriented (e.g., rotated 90degrees or at other orientations) and the spatially relative descriptorsused herein may likewise be interpreted accordingly.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments in accordance with thedisclosure; the methods, devices, and materials are now described. Allpatents and publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the materials andmethodologies that are reported in the publications may be used inconnection with the present disclosure.

The acronym “FET,” as used herein, refers to a field effect transistor.A type of FET is referred to as a “metal oxide semiconductor fieldeffect transistor” (MOSFET). MOSFETs can be planar structures built inand on the planar surface of a substrate such as a semiconductor wafer.MOSFETs can also have a three-dimensional, fin-based structures.

The term “bioFET” refers to a FET that includes a layer of capturereagents that act as surface receptors to detect the presence of atarget analyte of biological origin. A bioFET is a field-effect sensorwith a semiconductor transducer, according to some embodiments. Oneadvantage of bioFETs is the prospect of label-free operation.Specifically, bioFETs enable the avoidance of costly and time-consuminglabeling operations such as the labeling of an analyte with, forinstance, fluorescent or radioactive probes. One specific type of bioFETdescribed herein is a “dual-gate back-side sensing bioFET.” The analytesfor detection by a bioFET can be of biological origin such as, forexample and without limitation, proteins, carbohydrates, lipids, tissuefragments, or portions thereof. A bioFET can be part of a broader genusof FET sensors that may also detect a chemical compound; this type ofbioFET is known as a “ChemFET”) or any other element. A bioFET can alsodetect ions such as protons or metallic ions; this type of bioFET isknown as an “ISFET.” The present disclosure applies to all types ofFET-based sensors (“FET Sensors”). One specific type of FET Sensordescribed herein is a “Dual-Gate Back Side Sensing FET Sensor” (DG BSSFET Sensor).

“S/D” refers to the source/drain junctions that form two of the fourterminals of a FET.

The expression “high-k” refers to a high dielectric constant. In thefield of semiconductor device structures and manufacturing processes,high-k refers to a dielectric constant that is greater than thedielectric constant of SiO₂ (i.e., greater than 3.9).

The term “vertical,” as used herein, means nominally perpendicular tothe surface of a substrate.

The term “analysis” generally refers to a process or step involvingphysical, chemical, biochemical, or biological analysis that includes,but is not limited to, characterization, testing, measurement,optimization, separation, synthesis, addition, filtration, dissolution,or mixing.

The term “assay” generally refers to a process or step involving theanalysis of a chemical or a target analyte and includes, but is notlimited to, cell-based assays, biochemical assays, high-throughputassays and screening, diagnostic assays, pH determination, nucleic acidhybridization assays, polymerase activity assays, nucleic acid andprotein sequencing, immunoassays (e.g., antibody-antigen binding assays,ELISAs, and iqPCR), bisulfite methylation assays for detectingmethylation pattern of genes, protein assays, protein binding assays(e.g., protein-protein, protein-nucleic acid, and protein-ligand bindingassays), enzymatic assays, coupled enzymatic assays, kineticmeasurements (e.g., kinetics of protein folding and enzymatic reactionkinetics), enzyme inhibitor and activator screening, chemiluminescenceand electrochemiluminescence assays, fluorescent assays, fluorescencepolarization and anisotropy assays, absorbance and colorimetric assays(e.g., Bradford assay, Lowry assay, Hartree-Lowry assay, Biuret assay,and BCA assay), chemical assays (e.g., for the detection ofenvironmental pollutants and contaminants, nanoparticles, or polymers),and drug discovery assays, whole genome analysis, genome typinganalysis, genomic, exome analysis, micro-biome analysis, and clinicalanalysis including, but not limited to, cancer analysis, non-invasiveprenatal testing (NIPT) analysis, and/or UCS analysis. The apparatus,systems, and methods described herein may use or adopt one or more ofthese assays to be used with any of the FET Sensor described designs.

The term “liquid biopsy” generally refers to a biopsy sample obtainedfrom a subject's bodily fluid as compared to a subject's tissue sample.The ability to perform assays using a body fluid sample is oftentimesmore desirable than using a tissue sample. The less invasive approachusing a body fluid sample has wide ranging implications in terms ofpatient welfare, the ability to conduct longitudinal disease monitoring,and the ability to obtain expression profiles even when tissue cells arenot easily accessible, for example, in the prostate gland. Assays usedto detect target analytes in liquid biopsy samples include, but are notlimited to, those described above. As a non-limiting example, acirculating tumor cell (CTC) assay can be conducted on a liquid biopsysample.

For example, a capture reagent (e.g., an antibody) immobilized on a FETSensor may be used for detection of a target analyte (e.g., a tumor cellmarker) in a liquid biopsy sample using a CTC assay. CTCs are cells thathave shed into the vasculature from a tumor and circulate, for example,in the bloodstream. Generally CTCs are present in circulation in lowconcentrations. To assay the CTCs, CTCs are enriched from patient bloodor plasma by various techniques known in the art. CTCs may be stainedfor specific markers using methods known in the art including, but notlimited to, cytometry (e.g., flow cytometry)-based methods and IHC-basedmethods. For the apparatus, systems, and methods described herein, CTCsmay be captured or detected using a capture reagent or the nucleicacids, proteins, or other cellular milieu from the CTCs may be targetedas target analytes for binding to or detection by a capture reagent.

When a target analyte is detected on or from a CTC, for example, anincrease in target analyte expressing or containing CTCs may helpidentify the subject as having a cancer that is likely to respond to aspecific therapy (e.g., one associated with a target analyte) or allowfor optimization of a therapeutic regimen with, for example, an antibodyto the target analyte. CTC measurement and quantitation can provideinformation on, for example, the stage of tumor, response to therapy,disease progression, or a combination thereof. The information obtainedfrom detecting the target analyte on the CTC can be used, for example,as a prognostic, predictive, or pharmacodynamic biomarker. In addition,CTCs assays for a liquid biopsy sample may be used either alone or incombination with additional tumor marker analysis of solid biopsysamples.

The term “identification” generally refers to the process of determiningthe identity of a target analyte based on its binding to a capturereagent whose identity is known.

The term “measurement” generally refers to the process of determiningthe amount, quantity, quality, or property of a target analyte based onits binding to a capture reagent.

The term “quantitation” generally refers to the process of determiningthe quantity or concentration of a target analyte based on its bindingto a capture reagent.

The term “detection” generally refers to the process of determining thepresence or absence of a target analyte based on its binding to acapture reagent. Detection includes but is not limited toidentification, measurement, and quantitation.

The term “chemical” refers to a substance, compound, mixture, solution,emulsion, dispersion, molecule, ion, dimer, macromolecule such as apolymer or protein, biomolecule, precipitate, crystal, chemical moietyor group, particle, nanoparticle, reagent, reaction product, solvent, orfluid any one of which may exist in the solid, liquid, or gaseous state,and which can be the subject of an analysis.

The term “reaction” refers to a physical, chemical, biochemical, orbiological transformation that involves at least one chemical and thatgenerally involves (in the case of chemical, biochemical, and biologicaltransformations) the breaking or formation of one or more bonds such ascovalent, noncovalent, van der Waals, hydrogen, or ionic bonds. The termincludes chemical reactions such as, for example, synthesis reactions,neutralization reactions, decomposition reactions, displacementreactions, reduction-oxidation reactions, precipitation,crystallization, combustion reactions, and polymerization reactions, aswell as covalent and noncovalent binding, phase change, color change,phase formation, crystallization, dissolution, light emission, changesof light absorption or emissive properties, temperature change or heatabsorption or emission, conformational change, and folding or unfoldingof a macromolecule such as a protein.

“Capture reagent” as used herein, is a molecule or compound capable ofbinding the target analyte, which can be directly or indirectly attachedto a substantially solid material. The capture reagent can be achemical, and specifically any substance for which there exists anaturally occurring target analyte (e.g., an antibody, polypeptide, DNA,RNA, cell, virus, etc.) or for which a target analyte can be prepared,and the capture reagent can bind to one or more target analytes in anassay. The capture reagent may be non-naturally occurring ornaturally-occurring, and if naturally-occurring may be synthesized invivo or in vitro.

“Target analyte” as used herein, is the substance to be detected in thetest sample using embodiments of the present disclosure. The targetanalyte can be a chemical, and specifically any substance for whichthere exists a naturally occurring capture reagent (e.g., an antibody,polypeptide, DNA, RNA, cell, virus, etc.) or for which a capture reagentcan be prepared, and the target analyte can bind to one or more capturereagents in an assay. “Target analyte” also includes any antigenicsubstances, antibodies, and combinations thereof. The target analyte caninclude a protein, a peptide, an amino acid, a carbohydrate, a hormone,a steroid, a vitamin, a drug including those administered fortherapeutic purposes as well as those administered for illicit purposes,a bacterium, a virus, and metabolites of or antibodies to any of theabove substances.

“Test sample” as used herein, means the composition, solution,substance, gas, or liquid containing the target analyte to be detectedand assayed using embodiments of the present disclosure. The test samplecan contain other components besides the target analyte, can have thephysical attributes of a liquid, or a gas, and can be of any size orvolume, including for example, a moving stream of liquid or gas. Thetest sample can contain any substances other than the target analyte aslong as the other substances do not interfere with the binding of thetarget analyte with the capture reagent or the specific binding of thefirst binding member to the second binding member. Examples of testsamples include, but are not limited to, naturally-occurring andnon-naturally occurring samples or combinations thereof.Naturally-occurring test samples can be synthetic or synthesized.Naturally-occurring test samples include body or bodily fluids isolatedfrom anywhere in or on the body of a subject including, but not limitedto, blood, plasma, serum, urine, saliva or sputum, spinal fluid,cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluidof the respiratory, intestinal, and genitourinary tracts, tear fluid,saliva, breast milk, fluid from the lymphatic system, semen,cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cystfluid, amniotic fluid and combinations thereof, and environmentalsamples such as ground water or waste water, soil extracts, air, andpesticide residues or food-related samples.

Detected substances can include, for example, nucleic acids (includingDNA and RNA), hormones, different pathogens (including a biologicalagent that causes disease or illness to its host, such as a virus (e.g.,H7N9 or HIV), a protozoan (e.g., Plasmodium-causing malaria), or abacteria (e.g., E. coli or Mycobacterium tuberculosis), proteins,antibodies, various drugs or therapeutics or other chemical orbiological substances, including hydrogen or other ions, non-ionicmolecules or compounds, polysaccharides, small chemical compounds suchas chemical combinatorial library members, and the like. Detected ordetermined parameters may include but are not limited to, for example,pH changes, lactose changes, changing concentration, particles per unittime where a fluid flows over the device for a period of time to detectparticles, for example, particles that are sparse, and other parameters.

As used herein, the term “immobilized,” when used with respect to, forexample, a capture reagent, includes substantially attaching the capturereagent at a molecular level to a surface. For example, a capturereagent may be immobilized to a surface of the substrate material usingadsorption techniques including non-covalent interactions (e.g.,electrostatic forces, van der Waals, and dehydration of hydrophobicinterfaces) and covalent binding techniques where functional groups orlinkers facilitate attaching the capture reagent to the surface.Immobilizing a capture reagent to a surface of a substrate material maybe based upon the properties of the substrate surface, the mediumcarrying the capture reagent, and the properties of the capture reagent.In some cases, a substrate surface may be first modified to havefunctional groups bound to the surface. The functional groups may thenbind to biomolecules or biological or chemical substances to immobilizethem thereon.

The term “nucleic acid” generally refers to a set of nucleotidesconnected to each other via phosphodiester bond and refers to anaturally occurring nucleic acid to which a naturally occurringnucleotide existing in nature is connected, such as DNA includingdeoxyribonucleotides having any of adenine, guanine, cytosine, andthymine connected to each other and/or RNA including ribonucleotideshaving any of adenine, guanine, cytosine, and uracil connected to eachother. Naturally-occurring nucleic acids include, for example, DNA, RNA,and microRNA (miRNA). In addition, non-naturally occurring nucleotidesand non-naturally occurring nucleic acids are within the scope of thenucleic acids of the present disclosure. Examples include cDNA, peptidenucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), bridged nucleic acids/locked nucleic acids (BNA/LNA), andmorpholino nucleic acids. Further examples include chemically-modifiednucleic acids and nucleic acid analogues, such as methylphosphonateDNA/RNA, phosphorothioate DNA/RNA, phosphoramidate DNA/RNA, and2′-O-methyl DNA/RNA. Nucleic acids include those that may be modified.For example, a phosphoric acid group, a sugar, and/or a base in anucleic acid may be labeled as necessary. Any substances for nucleicacid labeling known in the art can be used for labeling. Examplesthereof include but are not limited to radioactive isotopes (e.g., 32P,3H, and 14C), DIG, biotin, fluorescent dyes (e.g., FITC, Texas, cy3,cy5, cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, and TAMRA), andluminescent substances (e.g., acridinium ester).

Aptamer as used herein refers to oligonucleic acids or peptide moleculesthat bind to a specific target molecule. The concept of usingsingle-stranded nucleic acids (aptamers) as affinity molecules forprotein binding was initially disclosed in Ellington, Andrew D., andJack W. Szostak, “Selection in vitro of single-stranded DNA moleculesthat fold into specific ligand-binding structures.” Nature 355 (1992):850-852; Tuerk, Craig, and Larry Gold, “Systematic evolution of ligandsby exponential enrichment: RNA ligands to bacteriophage T4 DNApolymerase.” Science 249.4968 (1990): 505-510) and is based on theability of short sequences to fold, in the presence of a target, intounique, three-dimensional structures that bind the target with highaffinity and specificity. Ng, Eugene W M, et al. “Pegaptanib, a targetedanti-VEGF aptamer for ocular vascular disease.” Nature Reviews, DrugDiscovery 5.2 (2006): 123, discloses that aptamers are oligonucleotideligands that are selected for high-affinity binding to moleculartargets.

The term “protein” generally refers to a set of amino acids linkedtogether usually in a specific sequence. A protein can be eithernaturally-occurring or non-naturally occurring. As used herein, the term“protein” includes amino acid sequences, as well as amino acid sequencesthat have been modified to contain moieties or groups such as sugars,polymers, metalloorganic groups, fluorescent or light-emitting groups,moieties or groups that enhance or participate in a process such asintramolecular or intermolecular electron transfer, moieties or groupsthat facilitate or induce a protein into assuming a particularconformation or series of conformations, moieties or groups that hinderor inhibit a protein from assuming a particular conformation or seriesof conformations, moieties or groups that induce, enhance, or inhibitprotein folding, or other moieties or groups that are incorporated intothe amino acid sequence and that are intended to modify the sequence'schemical, biochemical, or biological properties. As used herein,proteins include, but are not limited to, enzymes, structural elements,antibodies, antigen-binding antibody fragments, hormones, receptors,transcription factors, electron carriers, and other macromolecules thatare involved in processes such as cellular processes or activities.Proteins can have up to four structural levels that include primary,secondary, tertiary, and quaternary structures.

The term “antibody” as used herein refers to a polypeptide of theimmunoglobulin family that is capable of binding a corresponding antigennon-covalently, reversibly, and in a specific manner. For example, anaturally occurring IgG antibody is a tetramer including at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain includes a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. The heavychain constant region includes three domains, CHL CH2 and CH3. Eachlight chain includes a light chain variable region (abbreviated hereinas VL) and a light chain constant region. The light chain constantregion includes one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs arranged from amino-terminus to carboxy-terminusin the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Thethree CDRs constitute about 15-20% of the variable domains. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system. (Kuby,Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

The term “antibody” includes, but is not limited to, monoclonalantibodies, human antibodies, humanized antibodies, chimeric antibodies,and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Idantibodies to antibodies of the present disclosure). The antibodies canbe of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), orsubclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

The term “antigen binding fragment”, as used herein, refers to one ormore portions of an antibody that retain the ability to specificallyinteract with (e.g., by binding, steric hindrance,stabilizing/destabilizing, and spatial distribution) an epitope of anantigen. Examples of binding fragments include, but are not limited to,single-chain Fvs (scFv), camelid antibodies, disulfide-linked Fvs(sdFv), Fab fragments, F(ab′) fragments, a monovalent fragmentconsisting of the VL, VH, CL, and CH1 domains; a F(ab)2 fragment, abivalent fragment including two Fab fragments linked by a disulfidebridge at the hinge region; a Fd fragment consisting of the VH and CH1domains; a Fv fragment consisting of the VL and VH domains of a singlearm of an antibody; a dAb fragment (Ward, E. Sally, et al., “Bindingactivities of a repertoire of single immunoglobulin variable domainssecreted from Escherichia coli.” Nature 341.6242 (1989): 544-546), whichconsists of a VH domain; and an isolated complementarity determiningregion (CDR), or other epitope-binding fragments of an antibody.

Furthermore, although the two domains of the Fv fragment (VL and VH) arecoded for by separate genes, they can be joined (using recombinantmethods) by a synthetic linker that enables them to be made as a singleprotein chain, in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (“scFv”); see, e.g., Bird, RobertE., et al., “Single-chain antigen-binding proteins.” Science 242.4877(1988): 423-427; and Huston, James S., et al., “Protein engineering ofantibody binding sites: recovery of specific activity in an anti-digoxinsingle-chain Fv analogue produced in Escherichia coli.” Proceedings ofthe National Academy of Sciences 85.16 (1988): 5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen binding fragment.” These antigen binding fragments are obtainedusing conventional techniques known to those of skill in the art, andthe fragments are screened for utility in the same manner as are intactantibodies.

Antigen binding fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, single domain antibodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv(see, e.g., Holliger, Philipp, and Peter J. Hudson, “Engineered antibodyfragments and the rise of single domains.” Nature Biotechnology 23.9(2005): 1126). Antigen binding fragments can be grafted into scaffoldsbased on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat.No. 6,703,199, which describes fibronectin polypeptide monobodies).

Antigen binding fragments can be incorporated into single chainmolecules including a pair of tandem Fv segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions (Zapata, Gerardo, et al., “Engineering linearF(ab′)2 fragments for efficient production in Escherichia coli andenhanced antiproliferative activity.” Protein Engineering, Design andSelection 8.10 (1995): 1057-1062 and U.S. Pat. No. 5,641,870).

The term “monoclonal antibody” or “monoclonal antibody composition” asused herein refers to polypeptides, including antibodies and antigenbinding fragments that have substantially identical amino acid sequenceor are derived from the same genetic source. This term also includespreparations of antibody molecules of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope.

The term “nanoparticles” refers to atomic, molecular or macromolecularparticles in the length scale, for example, of approximately 1 to 100nm. Novel and differentiating properties and functions of nanoparticlesare observed or developed at a critical length scale of matter such as,for example, under 100 nm. Nanoparticles may be used in constructingnanoscale structures and may be integrated into larger materialcomponents, systems, and architectures. In some embodiments, thecritical length scale for novel properties and phenomena involvingnanoparticles may be under 1 nm (e.g., manipulation of atoms atapproximately 0.1 nm) or it may be larger than 100 nm (e.g.,nanoparticle reinforced polymers have the unique feature atapproximately 200 to 300 nm as a function of the local bridges or bondsbetween the nanoparticles and the polymer).

The term “nucleation composition” refers to a substance or mixture thatincludes one or more nuclei capable of growing into a crystal underconditions suitable for crystal formation. A nucleation composition may,for example, be induced to undergo crystallization by evaporation,changes in reagent concentration, adding a substance such as aprecipitant, seeding with a solid material, mechanical agitation, orscratching of a surface in contact with the nucleation composition.

The term “particulate” refers to a cluster or agglomeration of particlessuch as atoms, molecules, ions, dimers, polymers, or biomolecules.Particulates may include solid matter or be substantially solid, butthey may also be porous or partially hollow. They may contain a liquidor gas. In addition, particulates may be homogeneous or heterogeneous;that is, they may include one or more substances or materials.

The term “polymer” means any substance or compound that is composed oftwo or more building blocks (‘mers’) that are repetitively linked toeach other. For example, a “dimer” is a compound in which two buildingblocks have been joined together. Polymers include both condensation andaddition polymers. Examples of condensation polymers include polyamide,polyester, protein, wool, silk, polyurethane, cellulose, andpolysiloxane. Examples of addition polymers are polyethylene,polyisobutylene, polyacrylonitrile, poly(vinyl chloride), andpolystyrene. Other examples include polymers having enhanced electricalor optical properties (e.g., a nonlinear optical property) such aselectroconductive or photorefractive polymers. Polymers include bothlinear and branched polymers.

Overview of Exemplary Biosensing Device

FIG. 1 illustrates an overview of components that may be included in abiosensor system 100. Biosensor system 100 includes a sensor array 102having at least one sensing element for detecting a biological orchemical analyte and a fluid delivery system 104 designed to deliver oneor more fluid samples to sensor array 102. Fluid delivery system 104 maybe a microfluidic well positioned above sensor array 102 to contain afluid over sensor array 102. Fluid delivery system 104 may also includemicrofluidic channels for delivering various fluids to sensor array 102.Fluid delivery system 104 may include any number of valves, pumps,chambers, channels designed to deliver fluid to sensor array 102. Sensorarray 102 may include a repeating layout of sensors across a surface.For example, sensors may be arranged in a two-dimensional array of rowsand columns across the surface.

A readout circuit 106 is provided to measure signals from the sensors insensor array 102 and to generate a quantifiable sensor signal indicativeof the amount of a certain analyte that is present in a target solution,according to some embodiments.

A controller 108 may be used to send and receive electrical signals toboth sensor array 102 and readout circuit 106 to perform bio- orchemical-sensing measurements. Controller 108 may also be used to sendelectrical signals to fluid delivery system 104 to, for example, actuateone or more valves, pumps, or motors.

Sensor array 102 may include an array of bioFETs, where one or more ofthe bioFETs in the array are functionalized to detect a particulartarget analyte. Different ones of the sensors may be functionalizedusing different capture reagents for detecting different targetanalytes. Further details regarding an example design of particularbioFETs are provided below. The bioFETs may be arranged in a pluralityof rows and columns, forming a 2-dimensional array of sensors. In someembodiments, each row of bioFETs is functionalized using a differentcapture reagent. In some embodiments, each column of bioFETs isfunctionalized using a different capture reagent. In some embodiments,different sectors of the 2-dimensional array are functionalized withdifferent capture reagents.

Controller 108 may include one or more processing devices, such as amicroprocessor, and may be programmable to control the operation ofreadout circuit 106 and/or sensor array 102. The details of controller108 itself are not important for the understanding of the embodimentsdescribed herein. However, the various electrical signals that may besent and received from sensor array 102 will be discussed in more detailbelow.

Dual Gate Back-side FET Sensors

Embodiments described herein relate to measuring signals from one ormore bioFET sensors, or arrays of bioFET sensors, in a differentialmanner to reduce common noise between the bioFET sensors. Accomplishingthis goal involves controlling the fluid delivery to two separate bioFETsensors, or arrays of bioFET sensors, and differentially reading out themeasured signals from each of the bioFET sensors, or arrays of bioFETsensors. This particular section describes an example bioFET sensordesign that may be used in the embodiments of the present application.

One example type of bioFET sensor that may be used in sensor array 102is the dual gate back-side FET sensor. Dual gate back-side FET sensorsutilize semiconductor manufacturing techniques and biological capturereagents to form arrayed sensors. While MOSFETs can have a single gateelectrode connected to a single electrical node, the dual gate back-sidesensing FET sensor has two gate electrodes, each of which is connectedto a different electrical node. A first one of the two gate electrodesis referred to herein as a “front-side gate,” and the second one of thetwo gate electrodes is referred to herein as a “back-side gate.” Boththe front-side gate and the back-side gate are configured such that, inoperation, each one may be electrically charged and/or discharged andthereby each influences the electric field between the source/drainterminals of the dual gate back-side sensing FET sensor. The front-sidegate is electrically conductive, separated from a channel region by afront-side gate dielectric, and configured to be charged and dischargedby an electrical circuit to which it is coupled. The back-side gate isseparated from the channel region by a back-side gate dielectric andincludes a bio-functionalized sensing layer disposed on the back-sidegate dielectric. The amount of electric charge on the back-side gate isa function of whether a bio-recognition reaction has occurred. In theoperation of dual gate back-side sensing FET sensors, the front-sidegate is charged to a voltage within a predetermined range of voltages.The voltage on the front-side gate determines a correspondingconductivity of the FET sensor's channel region. A relatively smallamount of change to the electric charge on the back-side gate changesthe conductivity of the channel region. It is this change inconductivity that indicates a bio-recognition reaction.

One advantage of FET sensors is the prospect of label-free operation.Specifically, FET sensors enable the avoidance of costly andtime-consuming labeling operations such as the labeling of an analytewith, for instance, fluorescent or radioactive probes.

FIG. 2 illustrates an exemplary dual gate back-side sensing FET sensor200, according to some embodiments. Dual gate back-side sensing FETsensor 200 includes a control gate 202 formed on a surface of substrate214 and separated therefrom by an intervening dielectric 215 disposed onsubstrate 214. An interconnect region 211 including a plurality ofinterconnect layers may be provided over one side of substrate 214.Substrate 214 includes a source region 204, a drain region 206, and achannel region 208 between source region 204 and drain region 206. Insome embodiments, substrate 214 has a thickness between about 100 nm andabout 130 nm. Gate 202, source region 204, drain region 206, and channelregion 208 may be formed using suitable CMOS process technology. Gate202, source region 204, drain region 206, and channel region 208 form aFET. An isolation layer 210 is disposed on the opposing side ofsubstrate 214 from gate 202. In some embodiments, isolation layer 210has a thickness of about 1 μm. In this disclosure the side of substrate214 over which gate 202 is disposed is referred to as the “front-side”of substrate 214. Similarly, the side of substrate 214 on whichisolation layer 210 is disposed is referred to as the “back-side.”

An opening 212 is provided in isolation layer 210. Opening 212 may besubstantially aligned with gate 202. In some embodiments, opening 212 islarger than gate 202 and may extend over multiple dual gate back-sidesensing FET sensors. An interface layer (not shown) may be disposed inopening 212 on the surface of channel region 208. The interface layermay be operable to provide an interface for positioning and immobilizingone or more receptors for detection of biomolecules or bio-entities.Further details regarding the interface layer are provided herein.

Dual gate back-side sensing FET sensor 200 includes electrical contacts216 and 218 to drain region 206 and source region 204, respectively. Afront-side gate contact 220 may be made to gate 202, while a back-sidegate contact 222 may be made to channel region 208. It should be notedthat back-side gate contact 222 does not need to physically contactsubstrate 214 or any interface layer over substrate 214. Thus, while aFET can use a gate contact to control conductance of the semiconductorbetween the source and drain (e.g., the channel), dual gate back-sidesensing FET sensor 200 allows receptors formed on a side opposing gate202 of the FET device to control the conductance, while gate 202provides another region to control the conductance. Therefore, dual gateback-side sensing FET sensor 200 may be used to detect one or morespecific biomolecules or bio-entities in the environment around and/orin opening 212, as discussed in more detail using various examplesherein.

Dual gate back-side sensing FET sensor 200 may be connected to:additional passive components such as resistors, capacitors, inductors,and/or fuses; other active components, including p-channel field effecttransistors (PFETs), n-channel field effect transistors (NFETs),metal-oxide-semiconductor field effect transistors (MOSFETs), highvoltage transistors, and/or high frequency transistors; other suitablecomponents; or combinations thereof. It is further understood thatadditional features can be added in dual gate back-side sensing FETsensor 200, and some of the features described can be replaced oreliminated, for additional embodiments of dual gate back-side sensingFET sensor 200.

FIG. 3 illustrates a schematic of a portion of an exemplary addressablearray 300 of bioFET sensors 304 connected to bit lines 306 and wordlines 308. It is noted that the terms bit lines and word lines are usedherein to indicate similarities to array construction in memory devices,however, there is no implication that memory devices or a storage arraynecessarily be included in the array. Addressable array 300 may havesimilarities to that employed in other semiconductor devices such asdynamic random access memory (DRAM) arrays. For example, dual gateback-side sensing FET sensor 200, described above with reference to FIG.2, may be formed in a position that a capacitor would be found in a DRAMarray. Schematic 300 is exemplary only and one would recognize otherconfigurations are possible.

BioFET sensors 304 may each be substantially similar to dual gateback-side sensing FET sensor 200 according to some embodiments. FETs 302are configured to provide an electrical connection between a drainterminal of bioFET sensor 304 and bit line 306. In this way, FETs 302are analogous to access transistors in a DRAM array. In someembodiments, bioFET sensors 304 are dual gate back-side sensing FETsensors and each include a sensing gate provided by a receptor materialdisposed on a dielectric layer overlying a FET channel region disposedat a reaction site, and a control gate provided by a gate electrode(e.g., polysilicon) disposed on a dielectric layer overlying the FETchannel region.

Addressable array 300 shows an array formation designed to detect smallsignal changes provided by biomolecules or bio-entities introduced tobioFET sensors 304. The arrayed format using bit lines 306 and wordlines 308 allows for a smaller number of input/output pads since commonterminals of different FETs in the same row or column are tied together.Amplifiers may be used to enhance the signal strength to improve thedetection ability of the device having the circuit arrangement ofschematic 300. In some embodiments, when voltage is applied toparticular word lines 308 and bit lines 306, the corresponding accesstransistors 302 will be turned ON (e.g., like a switch). When the gateof the associated bioFET sensor 304 (e.g., such as back-side gate 222 ofthe dual gate back-side sensing FET sensor 200) has its charge affectedby the bio-molecule presence, a threshold voltage of bioFET sensor 304is changed, thereby modulating the current (e.g., I_(ds)) for a givenvoltage applied to back-side gate 222. The change of the current (e.g.,I_(ds)) or threshold voltage (V_(t)) can serve to indicate detection ofthe relevant biomolecules or bio-entities.

Referring to FIG. 4, an exemplary schematic 400 is presented. Exemplaryschematic 400 includes access transistor 302 and bioFET sensor 304arranged as an array 401 of individually addressable pixels 402. Array401 may include any number of pixels 402. For example, array 401 mayinclude 128×128 pixels. Other arrangements may include 256×256 pixels ornon-square arrays such as 128×256 pixels.

Each pixel 402 includes access transistor 302 and bioFET sensor 304along with other components that may include one or more heaters 408 anda temperature sensor 410. In this example, access transistor 302 is ann-channel FET. An n-channel FET 412 may also act as an access transistorfor temperature sensor 410. In some embodiments, the gates of FETs 302and 412 are connected, though this is not required. Each pixel 402 (andits associated components) may be individually addressed using columndecoder 404 and row decoder 406. In some embodiments, each pixel 402 hasa size of about 10 micrometers by about 10 micrometers. In someembodiments, each pixel 402 has a size of about 5 micrometers by about 5micrometers or has a size of about 2 micrometers by about 2 micrometers.

Column decoder 406 and row decoder 404 may be used to control the ON/OFFstate of both n-channel FETs 302 and 412 (e.g., voltage is applied tothe gates of FETs 302 and 412 together, and voltage is applied to thedrain regions of FETs 302 and 412 together). Turning ON n-channel FET302 provides a voltage to an S/D region of bioFET sensor 304. WhenbioFET sensor 304 is ON, a current I_(ds) flows through bioFET sensor304 and may be measured.

Heater 408 may be used to locally increase a temperature around bioFETsensor 304. Heater 408 may be constructed using any known technique,such as forming a metal pattern with a high current running through it.Heater 408 may also be a thermoelectric heater/cooler, like a Peltierdevice. Heater 408 may be used during certain biological tests such asto denature DNA or RNA or to provide a binding environment for certainbiomolecules. Temperature sensor 410 may be used to measure the localtemperature around bioFET sensor 304. In some embodiments, a controlloop may be created to control the temperature using heater 408 and thefeedback received from temperature sensor 410. In some embodiments,heater 408 may be a thermoelectric heater/cooler that allows for localactive cooling of the components within pixel 402.

Referring to FIG. 5, a cross section of an example dual gate back-sidesensing FET sensor 500 is provided, according to some embodiments. Thedual gate back-side sensing FET sensor 500 is one implementation of dualgate back-side sensing FET sensor 200. Thus previously describedelements from FIG. 2 are labeled with element numbers from FIG. 2 andtheir descriptions are not repeated here. Dual gate back-side sensingFET sensor 500 includes gate 202, source region 204, drain region 206,and channel region 208, where source region 204 and drain region 206 areformed within substrate 214. Gate 202, source region 204, drain region206, and channel region 208 form a FET. It should be noted that thevarious components of FIG. 3A are not intended to be drawn to scale andare exaggerated for visual convenience, as would be understood by aperson skilled in the relevant art.

In some embodiments, dual gate back-side sensing FET sensor 500 iscoupled to various layers of metal interconnects 502 that makeelectrical connection with the various doped regions and other devicesformed within substrate 214. Metal interconnects 502 may be manufacturedusing fabrication processes well known to a person skilled in therelevant art.

Dual gate back-side FET sensor 500 may include a body region 504separate from source region 204 and drain region 206. Body region 504may be used to bias the carrier concentration in channel region 208between source region 204 and drain region 206. In some embodiments, anegative voltage bias may be applied to body region 504 to improve thesensitivity of dual gate back-side FET sensor 500. In some embodiments,body region 504 is electrically connected to source region 204. In someembodiments, body region 504 is electrically grounded.

Dual gate back-side FET sensor 500 may be coupled to additionalcircuitry 506 fabricated within substrate 214. Circuitry 506 may includeany number of MOSFET devices, resistors, capacitors, and/or inductors toform circuitry to aid in the operation of dual gate back-side sensingFET sensor 500. Circuitry 506 may represent a readout circuit used tomeasure a signal from dual gate back-side FET sensor 500 that isindicative of analyte detection. Circuitry 506 may include amplifiers,analog to digital converters (ADCs), digital to analog converters(DACs), voltage generators, logic circuitry, and/or DRAM memory, to namea few examples. In some embodiments, circuitry 506 includes digitalcomponents and does not measure an analog signal from dual gateback-side FET sensor 500. All or some of the components of additionalcircuitry 506 may be integrated in the same substrate 214 as dual gateback-side FET sensor 500. It should be understood that many FET sensors,each substantially similar to dual gate back-side FET sensor 500, may beintegrated in substrate 214 and coupled to additional circuitry 506. Inanother example, all or some of the components of additional circuitry506 are provided on another semiconductor substrate separate fromsubstrate 214. In yet another example, some components of additionalcircuitry 506 are integrated in the same substrate 214 as dual gateback-side FET sensor 500, and some components of additional circuitry506 are provided on another semiconductor substrate separate fromsubstrate 214.

Still referring to the illustrative example of FIG. 5, dual gateback-side sensing FET sensor 500 includes an interface layer 508deposited over isolation layer 210 and within the opening over channelregion 208. In some embodiments, interface layer 508 has a thicknessbetween about 20 Å and about 40 Å. Interface layer 508 may be a high-Kdielectric material, such as hafnium silicate, hafnium oxide, zirconiumoxide, aluminum oxide, tantalum pentoxide, hafnium dioxide-alumina(HfO₂—Al₂O₃) alloy, or any combinations thereof. Interface layer 508 mayact as a support for the attachment of capture reagents as will bediscussed in more detail later in the section directed to biologicalsensing. A solution 512 is provided over the reaction site of dual gateback-side sensing FET sensor 500, and a fluid gate 510 is placed withinsolution 512. Solution 512 may be a buffer solution containing capturereagents, target analytes, wash solution, or any other biological orchemical species.

An example operation of dual gate back-side FET sensor 500 as a pHsensor will now be described with reference to FIG. 5.

Briefly, a fluid gate 510 is used to provide an electrical contact tothe “back gate” of dual gate back-side FET sensor 500. A solution 512 isprovided over the reaction site of dual gate back-side FET sensor 500,and fluid gate 510 is placed within solution 512. The pH of the solutionis generally related to the concentration of hydrogen ions [H⁺] in thesolution. The accumulation of the ions near the surface of interfacelayer 508 above channel region 208 affects the formation of theinversion layer within channel region 208 that forms the conductivepathway between S/D regions 204 and 206. In some embodiments, a currentI_(ds) flows from one S/D region to the other.

The current I_(ds) may be measured to determine the pH of solution 512.In some embodiments, fluid gate 510 is used as the gate of thetransistor during sensing while gate 202 remains floating. In someembodiments, fluid gate 510 is used as the gate of the transistor duringsensing while gate 202 is biased at a given potential. For example, gate202 may be biased at a potential between −2V and 2V depending on theapplication, while fluid gate 510 is swept between a range of voltages.In some embodiments, fluid gate 510 is biased at a given potential (orgrounded) while gate 202 is used as the gate of the transistor (e.g.,its voltage is swept across a range of potentials) during sensing. Fluidgate 510 may be formed from platinum or may be formed from any othercommonly used material(s) for reference electrodes in electrochemicalanalysis. An example of a reference electrode is a silver/silverchloride (Ag/AgCl) electrode, which has a stable potential value ofabout 0.230 V.

FIG. 6A shows ions in solution binding to a surface of interface layer508. A top-most atomic layer of interface layer 508 is depicted as thevarious dangling [O], [OH], and [OH₂ ⁺] bonds. As the ions accumulate onthe surface, the total surface charge affects the threshold voltage ofthe transistor. As used herein, the threshold voltage is the minimumpotential between the gate and the source of a FET sensor that isrequired to form a conductive path of minority carriers between thesource and the drain of the FET sensor. The total charge also directlyrelates to a pH of the solution, as a higher accumulation of positivecharge signifies a lower pH while a higher accumulation of negativecharge signifies a higher pH.

FIG. 6B illustrates an example change in threshold voltage that resultsdue to different pH values in an n-channel FET sensor. As can beobserved in this example, a 59 mV increase in threshold voltage roughlysignifies an increase of one in the pH of the solution. In other words,one pH change results in total surface charge equivalent of 59 mV whenmeasured as the voltage required to turn ON the transistor.

Changing the threshold voltage of dual gate back-side FET sensor 500also changes a time it takes to form a conductive path between S/Dregions 204 and 206 for a given voltage input to either fluid gate 510or gate 202. This time delay in “turning ON” the FET sensor may bequantified using digital circuitry and used to determine an analyteconcentration, according to some embodiments.

FIG. 7 illustrates an example biosensing test using dual gate back-sideFET sensor 500 to determine the local concentration of captured cells,according to some embodiments. Capture reagents 704 may be bound todielectric layer 508 using a linking molecule 702. Linking molecule 702may have a reactive chemical group that binds to a portion of dielectriclayer 508. An example of linking molecules include thiols. Linkingmolecules may also be formed via silanization of the surface ofdielectric layer 508, or by exposing the surface of dielectric layer 508to ammonia (NH₃) plasma, to form reactive NH₂ groups on the surface. Thesilanization process involves sequentially exposing the surface ofdielectric layer 508 to different chemicals to build up covalently-boundmolecules on the surface of dielectric layer 508, as would be generallyunderstood by a person skilled in the relevant art. Capture reagents 704may include antibodies that bind to proteins on the outer surface oftarget cells 706 to be captured.

According to some embodiments, target cells 706 produce chemicals thatmay alter the pH of the surrounding solution, which can be detected bybioFET sensor 500 as discussed above with reference to FIGS. 6A and 6B.In other embodiments, a solution 701 is introduced that includes asubstrate material, such as glucose, that is broken down by enzymeswithin target cells 706 to produce certain by-products. Examplesby-products may be acidic metabolic products produced by the glycolysisof glucose and the citric acid cycle (TCA). One specific example ofthese acidic metabolic products is poly(γ-glutamic acid), which ispositively charged, thus changing the pH of the surrounding solution andsignaling the presence of target cells 706. The entire chemical pathwaydiagramming the breakdown of glucose ultimately to producepoly(γ-glutamic acid) is illustrated in FIG. 8. It should be understoodthat other chemical pathways to produce other pH-altering by-productscan be used as well.

BioFET Array Embodiments

FIG. 9 illustrates a top-down view of an example sensor array 902 havinga plurality of bioFET sensors arranged in a repeating pattern. ThebioFET sensors arranged in sensor array 902 may each be examples of FETsensor 500 described above with reference to FIG. 5. A cross-sectionview taken across bioFET sensors 904 and 906 is illustrated in the upperleft of the figure. Although only a certain number of bioFET sensors areillustrated in sensor array 902, it should be understood that sensorarray 902 may include any number of bioFET sensors and that thearrangement of sensors is not limited to organized rows and columns.

Each of bioFET sensors 904 and 906 include corresponding wells 905 and907 that may be patterned by forming an opening through a thickness ofisolation layer 210. According to some embodiments, each well in sensorarray 902 is substantially aligned over a channel region of acorresponding dual gate back-side sensing FET sensor. In the illustratedexample, well 905 is aligned over channel region 208 a of bioFET sensor904 and well 907 is aligned over channel region 208 b of bioFET sensor906. According to some embodiments, each of the patterned wells acrosssensor array 906 has a size anywhere between 500 nm×500 nm and 500μm×500 μm. Sizes of the patterned wells between these dimensions canhelp to minimize a trade-off between effective sensing by each bioFETsensor and the number of different target analytes detected by sensorarray 902. According to some embodiments, the spacing between each ofthe wells across sensor array 906 is between 1 μm and 1 mm. Although notexplicitly illustrated in FIG. 9 for clarity, sensor array 902 may alsoinclude a microfluidic channel coupled to its surface, such that fluidcan be delivered via the microfluidic channel to each of the bioFETsensors in sensor array 902.

BioFET sensor 904 includes a dielectric layer 908 a patterned withinwell 905 and over channel region 208 a. BioFET sensor 906 similarlyincludes a dielectric layer 908 b patterned within well 907 and overchannel region 208 b. Dielectric layers 908 a and 908 b may be portionsof the same deposited dielectric layer, or may be layers having the samematerial composition but deposited at different times. In otherembodiments, dielectric layers 908 a and 908 b include differentmaterials.

Other components of sensor array 902 include a plurality of interconnectlayers (not shown) to make electrical connection to source/drain regions204/206 and gates of each of the bioFET sensors in the array. In theillustrated example, gates 202 a and 202 b are formed over a surface ofchannel region 208 a and 208 b, respectively. According to someembodiments, gates 202 a and 202 b are formed on a surface of channelregion 208 a and 208 b that is opposite to the surface of channel region208 a and 208 b having dielectric layer 908 a and 908 b. Any surfaces ofchannel region 208 a and 208 b should also be understood to be surfacesof semiconductor substrate 214. According to some embodiments, a carriersubstrate 612 is included to provide mechanical stability and stiffnessto sensor array 902.

In some embodiments, isolation regions 914 are formed between adjacentbioFET sensors to reduce electrical cross-talk between the sensors.Isolation regions 914 may represent standard shallow trench isolation(STI) structures filled with oxide.

Each bioFET sensor of sensor array 902 may be individually addressablesuch that sensing can occur independently at any of the bioFET sensorsin the array. In this way, multiple various analytes can be detectedusing the same sensor array 902 having different capture reagents boundto the dielectric layer of different bioFET sensors.

FIG. 10 illustrates sensor array 902 having some of the bioFET sensorsinclude capture reagents while other bioFET sensors do not. For example,bioFET sensor 1002 does not include any capture reagents while bioFETsensor 1004 includes capture reagents 1006 bound to its correspondingdielectric layer (as seen in the cross section taken across the lineA-A′). Any number of bioFET sensors in sensor array 902 can befunctionalized with capture reagents 1006, and similarly, any number ofbioFET sensors in sensor array 902 have no capture reagents. In someembodiments, the bioFET sensors having no capture reagents may be usedto provide control signals against those bioFET sensors that do havecapture reagents 1006. Capture reagents 1006 may be deposited overportions of sensor array 902 using various possible techniques, oneexample of which is described later with reference to FIG. 10.

Once capture reagents 1006 have been disposed on various bioFET sensors,a target solution containing a target analyte 1008 to be detected orcounted may be introduced over sensor array 902. For example, targetanalyte 1008 may include particular cells, like cancer cells, that bindto capture reagents 1006 as illustrated in the cross section B-B′ takenacross bioFET sensor 1004 after the target solution has been applied. Inother examples, target analyte 1008 includes any other type ofmicroorganism. The number, or density, of target analyte 1008 bound to aparticular bioFET sensor may be determined based on a change in themeasured drain current of the bioFET sensor. The exact sensingmethodology used for any given bioFET sensor is discussed later in moredetail with reference to FIGS. 14-15.

FIG. 11 illustrates an extension of the sensor array 902 illustrated inFIG. 10 by adding additional capture reagents bound to other bioFETsensors in the array. Specifically, bioFET sensor 1102 includes capturereagents 1104 that are different from capture reagents 1006 on bioFETsensor 1004 (as illustrated in FIG. 11.) Capture reagents 1104 may bedesigned to bind to different types of cells than capture reagents 1006.In other examples, capture reagents 1104 bind to any type of analytethat is different than an analyte that binds to capture reagents 1006.As illustrated in FIG. 11, a first plurality of bioFET sensors may befunctionalized using capture reagents 1006 while a second plurality ofbioFET sensors may be functionalized using capture reagents 1104.

According to an embodiment, a target solution is introduced over sensorarray 902 that contains various analytes (e.g., different cell types ordifferent microorganisms) which may bind either to capture reagents 1006of bioFET sensor 1004, capture reagents 1104 of bioFET sensor 1102, orto neither set of capture reagents. In the illustrated example, targetanalyte 1106 binds to capture reagent 1104. Target analyte 1106 mayinclude particular cells, like cancer cells, or any other type ofmicroorganism. The number, or density, of target analyte 1106 bound to aparticular bioFET sensor may be determined based on a change in themeasured drain current of the bioFET sensor. The exact sensingmethodology used for any given bioFET sensor is discussed later in moredetail with reference to FIGS. 14-15. Due to the individuallyaddressable nature of each bioFET sensor in sensor array 902, and theability to dispose different capture reagents on different bioFETsensors in the array, the detection of multiple different analytes canoccur simultaneously.

Detecting the presence of a given analyte may be used to provide abinary determination of whether or not the analyte is present in thetarget solution. In other embodiments, sensor array 902 is used toprovide a general count, or concentration, of a given analyte in atarget solution. In still other embodiments, sensor array 902 is used tomonitor the continual growth of cells or microorganisms captured over agiven bioFET sensor or plurality of bioFET sensors.

FIG. 12 illustrates sensor array 902 being used to monitor the growth ofcells or other microorganisms captured over given bioFET sensors in thearray, according to some embodiments. BioFET sensor 1202 is one exampleof a plurality of bioFET sensors in sensor array 902 that have captureda first population of cells 1204 shown in the cross-section A-A′ takenacross bioFET sensor 902. First population of cells 1204 may be capturedusing specific capture reagents present at one or more of the bioFETsensors in sensor array 902.

Culture media may be provided over sensor array 902 to allow firstpopulation of cells 1204 to grow into second population of cells 1206after a first time duration. Cross section B-B′ taken across bioFETsensor 902 illustrates the higher population of cells that make upsecond population of cells 1206. According to an embodiment, theincreased production of a positively charged by-product (such aspoly(γ-glutamic acid)) from each of second population of cells 1206 canbe detected by bioFET sensor 1202 as discussed above with reference toFIGS. 6-8. The measured increase in the drain current can be correlatedto a growth rate or total population size of the target cells.

At a later time period, second population of cells 1206 grows into ahigher third population of cells 1208 as shown in the cross-section C-C′take across bioFET sensor 1202. The same culture media may be used tocontinuously grow the cells from first population of cells 1204 to thirdpopulation of cells 1208. In another example, the culture media iscontinuously flown over sensor array 902 such that is remains fresh.According to an embodiment, the measured drain current of bioFET sensor1202 increases with a corresponding increase in the population size ofthe captured target cells. Depending on the type of cell being captured,the growth rate and corresponding change in the measured drain currentmay be different. Although this description focuses on the measurementof a single bioFET sensor 1202 in sensor array 902, it should beunderstood that multiple bioFET sensors of sensor array 902 may bemeasured together to provide a single signal indicative of the growthrate of the captured target cells at each of the multiple bioFETsensors. Furthermore, the growth rate of multiple different cell typescan be monitored using the same sensor array 902 by disposing differentcapture reagents on different sets of bioFET sensors.

In some embodiments, specific capture reagents are not used on thebioFET sensors of sensor array 902, and instead cells are disposed overthe surface of sensor array 902 and allowed to grow uninhibited over thesurface of sensor array 902. In this way, sensor array 902 can be usedto monitor how the cells spread across the surface of sensor array 902as they grow over time using the bioFET sensors.

FIG. 13 includes another top-down view of sensor array 902 andillustrates the deposition of different fluid droplets to immobilizedifferent capture reagents on different bioFET sensors, according tosome embodiments. For example, a first fluid droplet 1302 may bedeposited such that it covers a first plurality of bioFET sensors 1304.First fluid droplet 1302 may include a first plurality of capturereagents that bind to the dielectric layer of first plurality of bioFETsensors 1304. In one example, the first plurality of capture reagentsinclude antibodies designed to bind to a particular type of cell ormicroorganism. A second fluid droplet 1306 may be deposited such that itcovers a second plurality of bioFET sensors 1308. Second fluid droplet1306 may include a second plurality of capture reagents different fromthe first plurality of capture reagents. The second plurality of capturereagents bind to the dielectric layer of second plurality of bioFETsensors 1308. In one example, the second plurality of capture reagentsinclude antibodies designed to bind to another particular type of cellor microorganism different than that captured by first plurality ofcapture reagents. A third fluid droplet 1310 may be deposited such thatit covers a third plurality of bioFET sensors 1312. Third fluid droplet1310 may include a third plurality of capture reagents different fromthe first or second plurality of capture reagents. The third pluralityof capture reagents bind to the dielectric layer of third plurality ofbioFET sensors 1312. In one example, the third plurality of capturereagents include antibodies designed to bind to another particular typeof cell or microorganism different than that captured by either first orsecond plurality of capture reagents. Any number of droplets may be usedacross the surface of sensor array 902 to dispose different capturereagents across different sets of bioFET sensors.

According to some embodiments, each of droplets 1302, 1306, and 1310 aredeposited simultaneously across sensor array 902. In other embodiments,each of droplets 1302, 1306, and 1310 are disposed at different times.Each of droplets 1302, 1306, and 1310 may be left over theircorresponding bioFET sensors for a given period of time to ensure thatenough of the capture reagents bind to the bioFET sensors. Sensor array902 may be washed using a buffer solution between the deposition ofdifferent droplets. According to some embodiments, the diameter of anyone of droplets 1302, 1306, and 1310 is between 50 μm and 150 μm.

In some embodiments, the bioFET sensors of sensor array 902 can bearranged in other array configurations (e.g., staggered arrayconfiguration) instead of the linear array configuration shown in FIGS.9-13. In some embodiments, bioFET sensors in a staggered arrayconfiguration can be used to accommodate a larger number of bioFETsensors than a linear array of bioFET sensors within the same area. Insome embodiments, the bioFET sensors of sensor array 902 can be arrangedin a beehive configuration instead of the linear array configurationshown in FIGS. 9-13.

FIG. 14 illustrates an example method 1400 for using a sensor array todetect different target reagents, according to some embodiments. Each ofthe bioFET sensors in the sensor array may be a dual gate back-side FETsensor as illustrated in FIG. 5. It is understood that additionaloperations can be provided before, during, and after method 1400, andsome of the steps described below can be replaced or eliminated, foradditional embodiments of the method.

Method 1400 begins at block 1402 where a first fluid droplet isdeposited over the sensor array. The first fluid droplet includes afirst plurality of capture reagents, such as antibodies. The firstplurality of capture reagents bind to the backside surface of a firstplurality of bioFET sensors in the sensor array that are exposed to thefirst fluid droplet. The backside surface of each of the bioFET sensorsmay be within corresponding patterned wells across the surface of thesensor array. The first plurality of capture reagents in the first fluiddroplet may bind to a dielectric layer deposited on the backside surfaceof each of the first plurality of bioFET sensors. The first fluiddroplet may remain over the first plurality of bioFET sensors for agiven period of time to ensure sufficient binding of the capturereagents.

Method 1400 then proceeds to block 1404 where a second fluid droplet isdeposited over the sensor array. The second fluid droplet includes asecond plurality of capture reagents, such as antibodies, that aredifferent from the first plurality of capture reagents. The secondplurality of capture reagents bind to the backside surface of a secondplurality of bioFET sensors in the sensor array that are exposed to thesecond fluid droplet. The second plurality of bioFET sensors are eachdifferent than the first plurality of bioFET sensors such that thedifferent capture reagents are not bound to the same bioFET sensor. Thesecond plurality of capture reagents in the second fluid droplet maybind to a dielectric layer deposited on the backside surface of each ofthe second plurality of bioFET sensors. The second fluid droplet mayremain over the second plurality of bioFET sensors for a given period oftime to ensure sufficient binding of the capture reagents.

Method 1400 proceeds to block 1406 where target solution is providedover the sensor array, according to an embodiment. The target solutionmay be introduced by also dropping a droplet containing the targetsolution over various bioFET sensors in the sensor array. In anotherembodiment, the target solution is flown over the sensor array in amicrofluidic channel coupled to the surface of the sensor array, suchthat both the first plurality of bioFET sensors and the second pluralityof bioFET sensors in the sensor array are encompassed in the samemicrofluidic channel.

The target solution contains a plurality of target analytes, such astarget cells or microorganisms. The target analytes may bind to eitherthe first or second plurality of capture reagents to determine thepresence of the target analytes in the target solution. For example, themeasured drain current of corresponding bioFET sensors having thecaptured target analytes increases as the concentration of capturedtarget analytes increases. Since multiple different capture reagents maybe used across different bioFET sensors of the sensor array, multipledifferent target analytes can be sensed from a given target solution.

According to some embodiments, after the target analytes are bound totheir corresponding capture reagents in the sensor array, anothersolution containing a substrate material, such as glucose, is introducedover the sensor array. Captured cells and other types of microorganismscan break down the glucose to create positively charged by-products,which can be measured by the corresponding bioFET sensors as a change inthe drain current.

In block 1408, a first voltage is applied to the gates of the firstplurality of bioFET sensors and the induced drain current issubsequently measured from the first plurality of bioFET sensors.Similarly, in block 1410, a second voltage is applied to the gates ofthe second plurality of bioFET sensors and the induced drain current issubsequently measured from the second plurality of bioFET sensors. Thefirst and second applied voltages may have the same magnitude.

Method 1400 proceeds to block 1412 where a determination is made whetheror not the target analyte is present at either the first or secondplurality of bioFET sensors. As noted above, if the measured draincurrent of either the first or second plurality of bioFET sensorssignificantly increases above a baseline measurement (e.g., increasesbeyond changes caused by noise and standard measurement error), then itcan be determined that the target analyte is present at the bioFETsensors exhibiting the increased drain current. This determination maysimultaneously be made across all bioFET sensors in the sensor array totest for the presence of any number of target analytes.

FIG. 15 illustrates an example method 1500 for using a sensor array todetect the growth rate of target cells or microorganisms, according tosome embodiments. Each of the bioFET sensors in the sensor array may bea dual gate back-side FET sensor as illustrated in FIG. 5. It isunderstood that additional operations can be provided before, during,and after method 1500, and some of the steps described below can bereplaced or eliminated, for additional embodiments of the method.

Method 1500 begins at block 1502 where capture reagents are depositedover various bioFET sensors of a sensor array. The capture reagents maybe deposited in one or more droplets that are placed over the variousbioFET sensors, or flown across the various bioFET sensors in amicrofluidic channel. The capture reagents may include antibodiesdesigned to bind to a particular type of cell or microorganism.

After the capture reagents have had enough time to effectively bind tothe various bioFET sensors, the method proceeds to block 1504 where atarget solution having target cells or microorganisms is introduced overthe sensor array. The target solution may be introduced by also droppinga droplet containing the target solution over the various bioFET sensorsin the sensor array. In another embodiment, the target solution is flownover the sensor array in a microfluidic channel coupled to the surfaceof the sensor array. The cells or microorganisms present in the targetsolution bind to the capture reagents at the various bioFET sensors.

According to some embodiments, after the target cells or microorganismsare bound to their corresponding capture reagents in the sensor array,another solution containing a substrate material, such as glucose, isintroduced over the sensor array. The captured cells or microorganismscan break down the glucose to create positively charged by-products,which can be measured by the corresponding bioFET sensors as a change inthe drain current.

Method 1500 proceeds to block 1506 where a first voltage is applied tothe gates of the various bioFET sensors having the captured cells ormicroorganisms. The application of the first voltage causes the bioFETsensors to turn on and conduct, providing a measurable drain current. Atblock 1508, a first drain current is measured from the various bioFETsensors either individually or collectively. According to someembodiments, the magnitude of the measured first drain currentcorresponds to the concentration of the cells or microorganisms presentat the bioFET sensors at the current time that the first voltage isapplied.

Method 1500 proceeds to block 1510 where a second voltage is applied tothe gates of the various bioFET sensors having the captured cells ormicroorganisms. The second voltage may have the same magnitude as thefirst voltage and is applied to the gates of the various bioFET sensorsat a later time than the first voltage. At block 1512, a second draincurrent is measured from the various bioFET sensors either individuallyor collectively due to the application of the second voltage. Due to thedifference in time between the applied first and second voltages, thegrowth of the cells or microorganisms will cause an increase in themeasured second drain current compared to the measured first draincurrent. The difference between the first and second drain current maybe compared at block 1514 to determine a growth characteristic of thecells or microorganisms at the various bioFET sensors.

In some embodiments, instead of applying separate voltages at differenttimes to the gates of the various bioFET sensors to monitor the growthof the cells or microorganisms, a single voltage is continuously appliedand the drain current is continuously measured. In this way, the variousbioFET sensors of the sensor array can provide real-time monitoring ofthe growth of the cells or microorganisms.

General Biological Applications

BioFETs of the present disclosure may be used to determine the presenceor absence of a target analyte. In some aspects, the bioFETs may detectand measure absolute or relative concentrations of one or more targetanalytes. The bioFETs may also be used to determine static and/ordynamic levels and/or concentrations of one or more target analytes,providing valuable information in connection with biological andchemical processes. The bioFETs may further be used to monitor enzymaticreactions and/or non-enzymatic interactions including, but not limitedto, binding. As an example, the bioFETs may be used to monitor enzymaticreactions in which substrates and/or reagents are consumed and/orreaction intermediates, byproducts, and/or products are generated. Anexample of a reaction that can be monitored using a bioFET of thepresent disclosure is nucleic acid synthesis to, for example, ascertainnucleic acid sequence.

Types of target analytes for use in the embodiments of the presentdisclosure may be of any nature provided there exists a capture reagentthat binds to it selectively and in some instances specifically. Targetanalytes may be present in the test sample or, for example, generatedfollowing contact of the test sample with the sensing layer of a dualgate back-side sensing bioFET or with other reagents in the solution incontact with the sensing layer of a dual gate back-side sensing bioFET.Thus, types of target analytes include, but are not limited to, hydrogenions (protons) or other ionic species, non-ionic molecules or compounds,metals, metal coordination compounds, nucleic acids, proteins, lipids,polysaccharides, and small chemical compounds such as sugars, drugs,pharmaceuticals, chemical combinatorial library compounds, and the like.Target analytes may be naturally occurring or may be synthesized in vivoor in vitro. Target analytes may indicate that a reaction or interactionhas occurred, or indicate the progression thereof. Target analytesmeasured by a bioFET according to the present disclosure are not,however, limited and may include any of a variety of biological orchemical substances that provide relevant information regarding abiological or chemical process (e.g., binding events such as nucleicacid hybridization and other nucleic acid interactions, protein-nucleicacid binding, protein-protein binding, antigen-antibody binding,receptor-ligand binding, enzyme-substrate binding, enzyme-inhibitorbinding, cell stimulation and/or triggering, interactions of cells ortissues with compounds such as pharmaceutical candidates, and the like).It is to be understood that the present disclosure further contemplatesdetection of target analytes in the absence of a receptor, for example,detection of PPi and Pi in the absence of PPi or Pi receptors. Anybinding or hybridization event that causes a change to thetransconductance of the dual gate back-side sensing bioFET changes thecurrent that flows from the drain to the source of the sensors describedherein and can be detected according to some embodiments.

For detection of various target analytes, the sensing surfaces of thedual gate back-side sensing bioFETs of the present disclosure may becoated with a capture reagent for the target analyte that bindsselectively to the target analyte of interest or in some instances to agenus of analytes to which the target analyte belongs. A capture reagentthat binds selectively to a target analyte is a molecule that bindspreferentially to that analyte (i.e., its binding affinity for thatanalyte is greater than its binding affinity for any other analyte).Binding affinities for the analyte of interest may be at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 6-fold, at least about 7-fold, at least about8-fold, at least about 9-fold fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold, at least about 25-fold, at leastabout 30-fold, at least about 40-fold, at least about 50-fold, at leastabout 100-fold, at least about 500-fold, or at least about 1000-foldmore than its binding affinity for any other analyte. In addition torelative binding affinity, the capture reagent has an absolute bindingaffinity that is sufficiently high to efficiently bind the targetanalyte of interest (i.e., it has a sufficient sensitivity). Capturereagents for use in the methods and systems of the present disclosuremay have binding affinities in the femtomolar, picomolar, nanomolar, ormicromolar ranges and may be reversible.

The capture reagent may be of any nature (e.g., a chemical, a nucleicacid, a peptide, a lipid, or a combination thereof). The presentdisclosure contemplates capture reagents that are ionophores, which bindselectively to an ionic species, whether anionic or cationic. In someembodiments, an ionophore is the capture reagent and the ion to which itbinds is the target analyte. Ionophores include art-recognized carrierionophores (i.e., small lipid-soluble molecules that bind to aparticular ion) derived from, for example, a microorganism. In someembodiments, the capture reagent is polysiloxane, valinomycin, orsalinomycin and the ion to which it binds is potassium. In someembodiments, the capture reagent is monensin, nystatin, or SQI-Pr, andthe ion to which it binds is sodium. And in other embodiments, thecapture reagent is ionomycin, calcimycine (A23187), or CA 1001 (ETH1001), and the ion to which it binds is calcium. In other aspects, thepresent disclosure contemplates capture reagents that bind to more thanone ion. For example, beauvericin can be used to detect calcium and/orbarium ions, nigericin can be used to detect potassium, hydrogen and/orlead ions, and gramicidin can be used to detect hydrogen, sodium, and/orpotassium ions.

Test samples may be from a naturally occurring source or may benon-naturally occurring. Naturally-occurring test samples include,without limitation, bodily fluids, cells, or tissues to be analyzed fordiagnostic, prognostic and/or therapeutic purposes. The test sample mayinclude any of cells, nucleic acids, proteins, sugars, lipids, and thelike. In various embodiments, test samples may include chemical orbiological libraries to be screened for the presence of agents withparticular structural or functional attributes. Samples may be a liquidor dissolved in a liquid and of small volume and, as such, are amenableto high-speed, high-density analysis such as analyte detection usingmicrofluidics.

Examples of bioFETs contemplated by various embodiments discussed hereininclude, but are not limited to, chemical FETS (chemFETs), ion sensitiveFETs (ISFETs), immunologic FETs (ImmunoFETs), genetic FETs (GenFETs orDNA-FETs), enzyme FETs (EnFETs), receptor FETs, cell-based FETs,cell-free FETs, and liquid biopsy FETs. Thus, the bioFETs describedherein can be used to detect target analytes with capture reagents and,as such, define the bioFET type that are not mutually exclusive. As anon-limiting example, a liquid biopsy FET may detect cell-free DNA andmay also be referred to as a cell-free FET or a DNA-FET. See, e.g.,Sakata et al. “Potentiometric Detection of Single NucleotidePolymorphism by Using a Genetic Field-effect transistor,” Chembiochem 6(2005): 703-10; Uslu et al. “Labelfree fully electronic nucleic aciddetection system based on a field-effect transistor device,” BiosensBioelectron 19 (2004): 1723-31; Sakurai et al. “Real-time monitoring ofDNA polymerase reactions by a micro ISFET pH sensor,” Anal Chem 64.17(1992): 1996-1997.

For example, some embodiments provide a method for detecting a nucleicacid that includes contacting probe nucleic acids bound to a surface ofa back-side sensing layer of a dual gate back-side sensing bioFET with asample and detecting binding of a nucleic acid from the sample to one ormore regions of the probe nucleic acids. Such a nucleic acid detectingbioFET may also be referred to as a GenFET or DNA-FET.

In other aspects, some embodiments provide a method for detecting aprotein that includes contacting probe protein molecules bound to asurface of a back-side sensing layer of a dual gate back-side sensingbioFET with a sample and detecting binding of a protein from the sampleto one or more regions of the probe protein molecules. GenFETs andDNA-FETs may be used to detect the protein.

In other aspects, some embodiments provide a method for detecting anucleic acid that includes contacting probe protein molecules bound to asurface of a back-side sensing layer of a dual gate back-side sensingbioFET with a sample and detecting binding of a nucleic acid from thesample to one or more regions of the probe protein molecules. In yetother aspects, some embodiments provide a method for detecting anantigen that includes contacting probe antibodies bound to a back-sidesensing layer of a dual gate back-side sensing bioFET with a sample anddetecting binding of an antigen from the sample to one or more regionsof the probe antibodies. Such protein or antibody binding bioFETs mayalso be referred to as ImmunoFETs.

In other aspects, some embodiments provide a method for detecting anenzyme substrate or inhibitor that includes contacting probe enzymesbound to a surface of a back-side sensing layer of a dual gate back-sidesensing bioFET with a sample and detecting binding of an entity from (orgeneration of an enzymatic product in) the sample to one or more regionsof the probe enzymes. In yet other aspects, some embodiments provide amethod for detecting an enzyme that includes contacting enzymesubstrates or inhibitors bound to a surface of a back-side sensing layerof a dual gate back-side sensing bioFET with a sample and detectingbinding of an entity from (or generation of an enzymatic product in) thesample to one or more of the enzyme substrates or inhibitors. Such anenzyme based bioFET may also be referred to as an EnFET.

In other aspects, some embodiments provide a method for detectingprotein-small molecule (e.g., organic compound) interactions thatincludes contacting small molecules bound to a surface of a back-sidesensing layer of a dual gate back-side sensing bioFET with a sample anddetecting binding of proteins from the sample to one or more regions ofthe probe small molecules. In yet other aspects, some embodimentsprovide a method for detecting nucleic acid-small-molecule (e.g.,organic compound) interactions that includes contacting small moleculesbound to a surface of a back-side sensing layer of a dual gate back-sidesensing bioFET with a sample and detecting binding of nucleic acids fromthe sample to one or more regions of the probe small molecules. Ineither detection method, the sample may include small molecules and thecapture reagents bound to the surface of the back-side sensing layer maybe either nucleic acids or proteins. In other aspects, the targetanalytes of interest are heavy metals and other environmentalpollutants, and/or the bioFET arrays are specifically configured todetect the presence of different pollutants. Such small molecule orchemical-sensing bioFETs may also be referred to as chemFETs.

In other aspects, some embodiments provide a method for detectinghydrogen ions and/or changes in H+ concentration (i.e., changes in pH).Such ion-sensing bioFETs may also be referred to as ISFETs.

The systems and methods described herein can also be used to aid in theidentification and treatment of disease. For example, some embodimentsprovide a method for identifying a sequence associated with a particulardisease or for identifying a sequence associated with a response to aparticular active ingredient or treatment or prophylactic agent thatincludes contacting a capture reagent (e.g., a nucleic acid probe) boundto a surface of a back-side sensing layer of a dual gate back-sidesensing bioFET with a sample, and detecting binding of nucleic acids(e.g., including a variant or lacking nucleic acids otherwise containedin a corresponding wild-type nucleic acid sequence) from the sample toone or more regions of the capture reagent. Such bioFETs may also bereferred to as GenFETs, DNA-FETs, or liquid biopsy FETs.

Further Applications

Several additional applications of the dual gate back-side sensingbioFETs described herein are contemplated. For example, the sensinglayer of a dual gate back-side sensing bioFET provides real-time,label-free quantification and analysis for a variety of biological,chemical, and other applications including, but not limited to, geneexpression analysis, comparative genome hybridization (CGH), array-basedexon enrichment processes, protein sequencing, tissue microarrays, andcell culture. In some embodiments, the dual gate back-side sensingbioFET may be used to screen samples including, but not limited to,bodily fluids and/or tissues such as blood, urine, saliva, CSF, orlavages or environmental samples such as water supply samples or airsamples, for the presence or absence of a substance. For example, thearrays may be used to determine the presence or absence of pathogens(e.g., food-borne or infectious pathogens) such as viruses, bacteria, orparasites based on target genomic, proteomic, and/or other elements. Thearrays may also be used to identify the presence or absence orcharacterize cancer cells or cells that are indicative of anothercondition or disorder, in a subject. Additional applications for use ofthe dual gate back-side sensing bioFETs described herein include thosedescribed in U.S. Pat. No. 8,349,167 (Gene expression analysis,comparative genome hybridization (CGH), array-based exon enrichmentprocesses); U.S. Pat. No. 8,682,592 (Non-Invasive Prenatal Diagnosis(NIP D), DNA/RNA contamination, SNP identification); U.S. Pat. No.9,096,899 (Method of amplifying and sequencing DNA within a flow cell isprovided); U.S. Pat. No. 9,340,830 (Analyzing a tumor sample); U.S. Pat.No. 9,329,173 (Automated system for testing for Salmonella entericabacteria); U.S. Pat. No. 9,341,529 (Method for manufacturing a pressuresensor); U.S. Pub. Appl. Nos. 2015/0353920; 2015/0355129 (Chemical andbiological substances detection in bodily fluid); 2016/0054312(Chemically differentiated sensor array for sample analysis);2016/0040245 (Identification and molecular characterization of the CTCsassociated with neuroendocrine prostate cancer (NEPC).

In some embodiments, the dual gate back-side sensing bioFETs may be usedto obtain single cell gene expression profiles from one or more cells ina cellular sample of interest, for example, in heterogeneous cellularsamples. Such samples often exhibit a high degree of variation in theirgene/biomarker expression levels (e.g., due to the cell cycle,environment, and stochastic mechanism of transcription/translation),even among individual cells that have the same phenotype. The dual gateback-side sensing bioFETs enable interrogation of the expression profileof each cell in the sample. In certain aspects, the subject methods forsingle-cell molecular profiling obviate the need for separating cells ofinterest from a heterogeneous cellular sample with individual profilingavailable at each dual gate back-side sensing bioFET. Direct molecularprofiling in heterogeneous cell samples is advantageous for clinicaldiagnostic and biomarker discovery applications. In certain aspects, thedual gate back-side sensing bioFETs are used in molecular profiling andcellular subtyping of heterogeneous original or enriched disease tissueand biological fluid samples, for example, biopsy tumor samples,endothelial cells from cardiovascular disease samples, bone marrowsamples, lymph node samples, lymph, amniotic fluid, brain samples fromdifferent neurological disorders, lung pathological samples, and/or anyother heterogeneous disease tissue sample of interest. Thus, forexample, the dual gate back-side sensing bioFETs are used in themolecular profiling of normal biological tissue and biological fluidsamples, to elucidate, for example, the mechanisms of differentiation,immune responses, cell-cell communication, or brain development.

In some embodiments, the dual gate back-side sensing bioFETs are used inobtaining single cell expression profiles in circulating tumor cells(CTCs). CTCs may derive from metastases and can recirculate through thebloodstream and lymph to colonize distinct organs and/or the primarytumor, giving rise to secondary metastasis. CTCs play a critical role inthe metastatic spread of carcinomas. Therefore, detection of CTCs inblood (liquid biopsy) or disseminating tumor cells (DTC) in bone marrowmay be used to monitor tumor staging and would improve theidentification, diagnosis, and treatment of cancer patients at high riskof metastatic relapse. See, e.g., U.S. Pat. No. 9,340,830 (Col. 205,lines 61-64); U.S. Pat. No. 9,447,411 (Col. 21, lines 42-54); U.S. Pat.No. 9,212,977 (Col. 19, lines 56-67); U.S. Pat. No. 9,347,946 (Col. 9,lines 16-30). In some embodiments, the dual gate back-side sensingbioFETs are used to obtain expression and mutation profiles in acellular sample that includes CTCs as well as non-target contaminatingcell types (e.g., leukocytes). See, e.g., U.S. Pat. No. 9,340,830 (Col.1, lines 41-67); U.S. Pat. No. 9,447,411 (Col. 2, lines 41-55); U.S.Pat. No. 9,212,977 (Col. 2, lines 48-67; Col. 3 lines 1-10); and U.S.Pat. No. 9,347,946 (Col. 9).

In other embodiments, the dual gate back-side sensing bioFETs describedherein may provide point-of-care, portable, and/or real-time diagnostictools. They may, for example, provide an electronic readout of an enzymelinked immunosorbent assay (ELISA) or other assays to detect variouschemical or biological substances. The dual gate back-side sensingbioFETs may be configured to transduce or convert a biochemical bindingevent or reaction into an electrical signal, which may be read out.Indirect detection of a freely diffusing, electronically active speciesproduced at the site of a bound chemical or biological substance may beperformed utilizing the dual gate back-side sensing bioFETs. Electronicreadout ELISA schemes where an enzyme capable of producing anelectronically active species may be used. In some embodiments,riboswitches are used to detect metabolites. See, e.g., Mironov,Alexander S., et al., “Sensing small molecules by nascent RNA: amechanism to control transcription in bacteria.” Cell 111.5 (2002):747-756; Winkler, Wade, Ali Nahvi, and Ronald R. Breaker, “Thiaminederivatives bind messenger RNAs directly to regulate bacterial geneexpression.” Nature 419.6910 (2002): 952-956. In some embodiments, thedual gate back-side sensing bioFET arrays are used to measure thekinetics of a reaction and/or compare the activities of enzymes,including substrates, a co-factor, or another moiety for readout.

Other applications for the dual gate back-side sensing bioFET arraysinvolve the use of molecular recognition sites, where molecules thatspecifically recognize particular target molecules are either identifiedor designed and applied to the surface of the array. Previous work withchemFETs has demonstrated the ability of single individual ISFETs torecognize ions such as potassium.

In some embodiments, the dual gate back-side sensing bioFET is used tomonitor the presence and/or amount of specific molecules including, forexample, environmental testing of specific toxins and importantelements. Such testing may use molecular recognition sites to measureboth pollution gases and particulate contamination, where molecules thatspecifically recognize particular target molecules are either identifiedor designed and applied to the surface of the array. See, e.g., Brzozkaet al. “Enhanced performance of potassium CHEMFETs by optimization of apolysiloxane membrane,” Sensors and Actuators B. Chemical 18, 38-41(1994); Sibbald et al. “A miniature flow-through cell with afour-function ChemFET integrated circuit for simultaneous measurementsof potassium, hydrogen, calcium and sodium ions,” Analytica ChimicaActa. 159, 47-62 (1984); Cobben et al. “Transduction of selectiverecognition of heavy metal ions by chemically modified field effecttransistors (CHEMFETs),” Journal of the American Chemical Society 114,10573-10582 (1992). In some embodiments, the dual gate back-side sensingbioFET can be used with a personal, portable, and wearable detectorsystem. This system can act as an early warning device indicating to theuser that the pollution levels in their current local environment is ata level that could cause the user some discomfort or even lead tobreathing problems. This is particularly relevant to people sufferingfrom respiratory or bronchial or asthma conditions, where the user needsto take necessary precautions. The dual gate back-side sensing bioFEThas the capability of detecting individual gases such as, for example,NOx, SO₂ and or CO and/or monitoring temperature and humidity. See U.S.Pub. Appl. Nos. 2014/0361901; 2016/0116434 (Paragraph [0117]). Thepollution sensors may, for example, be referred to as a gas fieldeffective transistor (gasFET). A gasFET may contain, for example, an FETwith a gate metallization exposed to the surrounding atmosphere. When agas is absorbed on the surface, protons can diffuse to the metal gasinterface. This results in a dipole layer which affects the thresholdvoltage of the device.

In some embodiments, the dual gate back-side sensing bioFET may be usedin vivo by introduction into a subject (e.g., in the brain or otherregion that is subject to ion flux) and then analyzing for changes. Forexample, electrical activity of cells may be detected by ionic flow.Thus, a bioFET array can be integrated onto a novel ion-discriminatingtissue probe. Other applications include, for example, cochlearprosthesis and retinal and cortical implants. See, e.g., Humayun et al.Vision Research 43, 2573-2581 (2003); Normann et al. Vision Research 39,2577-2587 (1999).

Final Remarks

Described herein are embodiments of a sensor array having a plurality ofdual-gate back side sensing bioFETs and methods of using the sensorarray. According to some embodiments, a sensor array includes asemiconductor substrate, a first plurality of FET sensors and a secondplurality of FET sensors. The first plurality of FET sensors eachincludes a first channel region between a source and a drain region inthe semiconductor substrate and underlying a gate structure disposed ona first side of the first channel region, and a dielectric layerdisposed on a second side of the first channel region opposite from thefirst side of the first channel region. A first plurality of capturereagents is coupled to the dielectric layer over the first channelregion. The second plurality of FET sensors each includes a secondchannel region between a source and a drain region in the semiconductorsubstrate and underlying a gate structure disposed on a first side ofthe second channel region, and the dielectric layer disposed on a secondside of the second channel region opposite from the first side of thesecond channel region. A second plurality of capture reagents is coupledto the dielectric layer over the second channel region. The secondplurality of capture reagents is different from the first plurality ofcapture reagents. The first plurality of FET sensors and the secondplurality of FET sensors are arranged in a two-dimensional array.

According to some embodiments, a method of using a sensor deviceincludes depositing a first droplet of solution containing a firstplurality of capture reagents over a first plurality of FET sensorsformed in a semiconductor substrate. The first plurality of capturereagents binds to a dielectric layer on a first surface of thesemiconductor substrate in a first plurality of openings arranged overthe first plurality of FET sensors. The method also includes depositinga second droplet of solution containing a second plurality of capturereagents over a second plurality of FET sensors formed in thesemiconductor substrate. The second plurality of capture reagents bindsto the dielectric layer on the first surface of the semiconductorsubstrate in a second plurality of openings arranged over the secondplurality of FET sensors. The second plurality of capture reagents isdifferent than the first plurality of capture reagents. The method alsoincludes introducing a target solution over the first plurality of FETsensors and the second plurality of FET sensors. The method alsoincludes applying a first voltage to a plurality of first gatestructures of the first plurality of FET sensors. The first gatestructures are on a second surface of the semiconductor substrateopposite to the first surface of the semiconductor substrate. The methodalso includes applying a second voltage to a plurality of second gatestructures of the second plurality of FET sensors. The second gatestructures are on a second surface of the semiconductor substrateopposite to the first surface of the semiconductor substrate. The methodincludes determining the presence of one or more target analytes in thetarget solution based on the application of at least one of the firstvoltage or the second voltage.

According to some embodiments, a method of using a sensor deviceincludes depositing a solution containing a plurality of capturereagents over a plurality of FET sensors formed in a semiconductorsubstrate. The plurality of capture reagents binds to a dielectric layeron a first surface of the semiconductor substrate in a plurality ofopenings arranged over the plurality of FET sensors. The method alsoincludes introducing a second solution over the plurality of FET sensorssuch that one or more cells in the second solution binds to the capturereagents bound to the dielectric layer of the plurality of FET sensors.The method includes applying a first voltage to a plurality of gatestructures of the plurality of FET sensors. The plurality of gatestructures is on a second surface of the semiconductor substrateopposite to the first surface of the semiconductor substrate. A firstcurrent response of the plurality of FET sensors is measured based onthe applying of the first voltage. The method also includes applying asecond voltage to the plurality of gate structures of the plurality ofFET sensors at a given time period after the applying of the firstvoltage, and measuring a second current response of the plurality of FETsensors based on the applying of the second voltage. The method includesdetermining a growth characteristic of the one or more cells based on acomparison between the first current response and the second currentresponse.

It is to be appreciated that the Detailed Description section, and notthe Abstract of the Disclosure section, is intended to be used tointerpret the claims. The Abstract of the Disclosure section may setforth one or more but not all exemplary embodiments of the presentdisclosure as contemplated by the inventor(s), and thus, is not intendedto limit the present disclosure and the subjoined claims in any way.

It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments but should be definedin accordance with the subjoined claims and their equivalents.

What is claimed is:
 1. A method, comprising: depositing a first dropletof solution containing a first plurality of capture reagents over afirst plurality of FET sensors formed in a semiconductor substrate, suchthat the first plurality of capture reagents binds to a dielectric layeron a first surface of the semiconductor substrate in a first pluralityof openings arranged over the first plurality of FET sensors; depositinga second droplet of solution containing a second plurality of capturereagents over a second plurality of FET sensors formed in thesemiconductor substrate, such that the second plurality of capturereagents binds to the dielectric layer on the first surface of thesemiconductor substrate in a second plurality of openings arranged overthe second plurality of FET sensors, the second plurality of capturereagents being different than the first plurality of capture reagents;introducing, using a fluid delivery system, a target solution over thefirst plurality of FET sensors and the second plurality of FET sensors;coupling a plurality of first gate structures of the first plurality ofFET sensors to a controller configured to apply a first voltage to theplurality of first gate structures, wherein the plurality of first gatestructures is on a second surface of the semiconductor substrateopposite to the first surface of the semiconductor substrate; coupling aplurality of second gate structures of the second plurality of FETsensors to the controller configured to apply a second voltage to theplurality of second gate structures, wherein the plurality of secondgate structures is on a second surface of the semiconductor substrateopposite to the first surface of the semiconductor substrate; andcoupling the first and second pluralities of FET sensors to a readoutcircuit configured to determine the presence of one or more targetanalytes in the target solution based on the application of at least oneof the first voltage and the second voltage.
 2. The method of claim 1,wherein the introducing the target solution comprises flowing the targetsolution through a microfluidic channel of the fluid delivery system. 3.The method of claim 1, wherein the first droplet and the second dropleteach have a diameter around 100 μm.
 4. The method of claim 1, furthercomprising binding the one or more target analytes to either the firstplurality of capture reagents or the second plurality of capturereagents.
 5. The method of claim 4, further comprising introducing asolution having a compound that reacts with the one or more targetanalytes to produce a by-product.
 6. The method of claim 5, wherein apresence of the by-product adjacent to either the first plurality ofcapture reagents or the second plurality of capture reagents changes athreshold voltage of the first plurality of FET sensors or the secondplurality of FET sensors, respectively.
 7. A method of fabricating abiosensor system, the method comprising: depositing a solutioncontaining a plurality of capture reagents over a plurality of FETsensors formed in a semiconductor substrate, such that the plurality ofcapture reagents binds to a dielectric layer on a first surface of thesemiconductor substrate in a plurality of openings arranged over theplurality of FET sensors; introducing, using a fluid delivery system, asecond solution over the plurality of FET sensors such that one or morecells in the second solution binds to the capture reagents bound to thedielectric layer of the plurality of FET sensors; coupling a pluralityof gate structures of the plurality of FET sensors to a controllerconfigured to apply first and second voltages to the plurality of gatestructures, wherein the plurality of gate structures is on a secondsurface of the semiconductor substrate opposite to the first surface ofthe semiconductor substrate and wherein the second voltage is applied ata given time period after the applying of the first voltage; andcoupling the plurality of FET sensors to a readout circuit configured tomeasure first and second current responses of the plurality of FETsensors based on the applying of the first and second voltages, whereinthe readout circuit is further configured to determine a growthcharacteristic of the one or more cells based on a comparison betweenthe first and second current responses.
 8. The method of claim 7,wherein the introducing the target solution comprises flowing the targetsolution through a microfluidic channel of the fluid delivery system. 9.The method of claim 7, wherein the depositing comprises depositing thesolution containing the plurality of capture reagents as a droplet overthe plurality of FET sensors.
 10. The method of claim 7, furthercomprising binding the one or more cells to the plurality of capturereagents.
 11. The method of claim 10, further comprising introducing asolution having a compound that reacts with the one or more target cellsto produce a by-product.
 12. The method of claim 11, wherein a presenceof the by-product adjacent to the plurality of capture reagents changesa threshold voltage of the plurality of FET sensors.
 13. A method,comprising: forming a first plurality of FET sensors with first channelregions formed in a substrate and first gate structures formed on firstsides of the first channel regions; forming a second plurality of FETsensors with second channel regions formed in the substrate and secondgate structures formed on first sides of the second channel regions,wherein the first and second plurality of FET sensors are arranged in atwo-dimensional array; depositing a dielectric layer on second sides ofthe first and second channel regions, wherein the second sides areopposite to the first sides; coupling a first plurality of capturereagents to portions of the dielectric layer that are disposed on thesecond sides of the first channel regions; coupling a second pluralityof capture reagents to portions of the dielectric layer that aredisposed on the second sides of the second channel regions, wherein thesecond plurality of capture reagents is different from the firstplurality of capture reagents; and coupling the first and secondplurality of FET sensors to a common reference electrode.
 14. The methodof claim 13, further comprising depositing an insulating layer on afirst side of the substrate, wherein the first and second gatestructures are formed on a second side of the substrate that is oppositeto the first side of the substrate.
 15. The method of claim 14, furthercomprising forming a plurality of openings in the insulating layer,wherein the plurality of openings are formed on the first and secondchannel regions.
 16. The method of claim 15, wherein the depositing thedielectric layer comprises depositing the dielectric layer within theplurality of openings.
 17. The method of claim 15, wherein the formingthe plurality of openings comprises forming the plurality of openingswith an area between 500 nm×500 nm and 500 μm×500 μm.
 18. The method ofclaim 13, wherein the coupling the first and second plurality of capturereagents comprises coupling RNA, DNA, antibodies, enzymes, proteins, orcells.
 19. The method of claim 13, wherein the depositing the dielectriclayer comprises depositing a high-k dielectric material.
 20. The methodof claim 13, further comprising forming a microfluidic channel on thefirst and second plurality of FET sensors.